1. Interferons and Interferon Regulatory Factors 1 and 2
Interferons (IFNs) belong to a family of pleiotropic cytokines which were originally identified based on their anti-viral properties. A variety of tissues generate type I IFNs, i.e. IFN-.alpha.s and IFN-.beta., upon viral infection and the secreted IFNs subsequently exert their anti-viral activity on target cells by inducing a set of genes, the IFN-inducible genes. Recently, much attention has been focused on the role of IFNs in cell growth and differentiation, and it has been shown that IFNs exhibit anti-proliferative effects on many normal and transformed cells (reviewed by Weissmann and Weber, Prog. NucIeic Acid Res. Mol. Biol. 33: 251-300 (1986); Pestka et al., Annu. Rev. Biochem. 56: 727-777 (1987); De Maeyer and De Maeyer-Guignard, Interferons and Other Regulatory Cytokines, New York, John Wiley and Sons (1988); Taniguchi, Annu. Rev. Immunol. 6: 439-464 (1988); Vilcek, "Interferons etc.," Handbook of Experimental Pharmacology, Sporn and Roberts, eds., Berlin, Springer-Verlag, pp. 3-38 (1990); Sen and Lengyel, J. Biol. Chem. 267: 5017-5020 (1992)). In addition, a number of studies have demonstrated that IFNs and growth stimulatory factors act in a mutually antagonistic manner; IFNs have been shown to block growth factor-stimulated cell cycle transitions, while certain growth factors have been shown to reverse the anti-proliferative effects of IFNs. Furthermore, IFNs are induced by a number of growth factors, suggesting a physiological role for IFNs in a feedback mechanism that regulates cell growth. Hence these observations have lent support to the prevailing notion that IFNs are "negative growth factors" (reviewed by Clements and McNurian, Biochem. J. 226: 345-360 (1985); Tamm et al., Interferon 9, I. Gresser, ed., California, Academic Press, pp. 14-74 (1987); De Maeyer and De Maeyer-Guignard, ibid. (1988); Gresser, Acta Oncologica 28: 347-353 (1989); Vilcek, ibid. (1990)). In this context, it is interesting to note that type I IFN genes are frequently deleted in some types of malignancies (Diaz et al., Proc. Natl. Acad. Sci. USA 85: 5259-5263 (1988); Miyakoshi et al., Cancer Res. 50: 278-283 (1990)). However, little is known about the mechanistic aspects of these anti-proliferative effects of IFNs.
During studies on the regulatory mechanism(s) of human IFN-.beta. gene, two novel DNA-binding factors, Interferon Regulatory Factor-1 (IRF-1) and -2 (IRF-2) were identified (Fujita et al., EMBO J. 7: 3397-3405 (1988); Miyamoto et al., Cell 54: 903-913 (1988); Harada et al., Cell 58: 729-739 (1989)). The amino acid sequences for human and mouse IRF-1 and mouse IRF-2 as well as the DNA sequences coding therefore are also disclosed in the U.S. patent application Ser. No. 07/397,967, filed Aug. 24, 1989. These two factors are structurally related, particularly in the N-terminal regions which confer DNA binding specificity. In fact, both factors bind to the same DNA sequence elements found within the promoters of IFN-.alpha.s, IFN-.beta. and many IFN-inducible genes (Harada et al., ibid. (1989)). A series of gene transfection studies have demonstrated that IRF-1 functions as a critical activator for IFN and IFN-inducible genes, Whereas IRF-2 represses the IRF-1 effect (Fujita et al., Nature 337: 270-272 (1989); Harada et al., Cell 63: 303-312 (1990); Naf et al., Proc. Natl. Acad. Sci. USA 88: 1369-1373 (1991); Au et al., Nucl. Acids Res. 20: 2877-2884 (1992); Reis et al., EMBO J. 11: 185-193 (1992); Stark and Kerr, J. Interferon R. 12: 147-151 (1992)). In the context of the IFN-mediated cellular response, it is interesting that expression of IRF-1 gene itself is IFN-inducible. The IRF-2 gene is also induced in IFN-stimulated cells, but this induction occurs only following IRF-1 gene induction (Harada et at., ibid. (1989)). Moreover, previous studies have revealed that IRF-1 and IRF-2 differ in terms of their stability; the former has a short half-life (about 30 min.), whereas the latter is relatively stable (half-life; about 8 hrs) in INF-treated or virus-infected cells. In growing cells, IRF-2 levels are higher than those of IRF-1, but the IRF-1/IRF-2 ratio increases following stimulation by IFNs or viruses (Watanabe et at., Nucl. Acid Res. 19: 4421-4428 (1991)). Therefore, a transient increase in the IRF-1/IRF-2 ratio may be a critical event in the regulation of cell growth by IFNs. Consistent with this notion are the findings that transgenic mice carrying the human IRF-1 gene linked to the human immunoglobulin gene enhancer are deficient in developing B lymphocytes (Yamada et al., Proc. Natl. Acad. Sci. USA 88: 532-536 (1991)).
2. Tumor Suppressor Genes
Human tumorigenesis is a multistep process resulting from the progressive acquisition of mutations at multiple genetic loci that regulate cell growth, differentiation, and metastasis. In the best-studied human tumor models, "gain-of-function" mutations found in dominantly-acting proto-oncogenes are accompanied by "loss-of-function" mutations in tumor suppressor genes. Although numerous proto-oncogenes were initially identified and characterized, recent studies have identified several tumor suppressor genes whose mutation or deletion appears to be critical for the development of human tumors, including RB, p53, and WT1 (reviewed in Marshall, Cell 64: 313-326 (1991)), as well as APC (Groden et al., Cell 66: 589-600 (1991); Kinzler et al., Science 253: 661-664 (1991)), and NF1 (Xu et al., Cell 62: 599-608 (1990); Marshall, ibid. (1991); Li et al., Cell 69: 275-281 (1992)). The loss of heterozygosity at additional genetic loci (Ponder, Nature 335: 400-402 (1988); Marshall, ibid. (1991)) and the recurrent deletion of specific chromosomal regions in human tumors have supported the view that many more candidate tumor suppressor genes remain to be identified.
An interstitial deletion of the long arm of chromosome 5(del(5q); the "5q-" eytogenetic abnormality) or loss of a whole chromosome 5(-5 or monosomy 5) are among the most frequent recurrent cytogenetic abnormalities in human leukemia and the preleukemic myelodysplastic syndromes (myelodysplasia; MDS). Del(5q)or monosomy 5 is found in 30% of patients with MDS, in 50% of patients with secondary or therapy-induced acute myelogenous leukemia (AML), and in 15% and 2% of patients with de novo AML and de novo acute lymphocytic leukemia (ALL), respectively (Van den Berghe et al., Nature 251: 437 (1974), Cancer Genet. Cytogenet. 17: 189-255 (1985); Fourth International Workshop on Chromosomes in Leukemia, (1982); Le Beau et al., J. Clin. Oncol. 4: 325-345 (1986); Nimer and Golde, Blood 70: 1705-1712 (1987); Kerim et al. Leukemia 4: 12-15 (1990); Pederson-Bjergaard et al., Blood 76: 1083-1091 (1990)). The del(5q) was first described as the hallmark of a unique myelodysplastic syndrome (the "5q-Syndrome") occurring predominantly in elderly females that is characterized by refractory anemia, thrombocytosis, and abnormal megakaryocytes (Van den Berghe et al., ibid. (1974)). Females with this syndrome usually have an indolent clinical course; the affected myeloid stem cell clone appears to have a slow capacity for expansion, acquires additional cytogenetic abnormalities only infrequently, and transforms to AML in only 10-20% of cases (Van den Berghe et al., ibid. (1985); Dewald et al., Blood 66: 189-197 (1985); Nimer and Gold, ibid. (1987)). In contrast, patients who present with de novo or secondary AML with del(5q) usually have additional cytogenetic abnormalities at presentation and a very poor prognosis (Rowly et al., Blood 58: 759-767 (1981); Fourth International Workshop on Chromosomes in Leukemia (1982); Le Beau et al., ibid. (1986); Samuels et al., Leukemia 2: 79-83 (1988)). In AML, the presence of a del(5q)/-5 has also been associated with occupational exposure to carcinogens (Mitelman et al., Blood 52: 1229-1273 (1978); Golomb et at., Blood 60: 404-411 (1982)) or with previous exposure to alkylating agent chemotherapy or radiotherapy for the treatment of various malignancies (Le Beau et al., ibid. (1986)). A series of studies have revealed that the smallest commonly deleted segment of the del(5q), the so called "critical" region, lies in band 5q31 (Le Beau et al., Blood 73: 647-650 (1989); Pederson and Jensen, Leukemia 5: 566-573 (1991)). Rare de novo AMLs with translocations involving 5q31 have also been described (Fourth International Workshop on Chromosomes in Leukemia, 1982). These findings suggest that the causative gene(s) lies in 5q31 and that deletion of this gene(s) may be central to the pathogenesis of leukemia and MDS. Numerous candidate genes have been mapped to the 5q31 region, including the hematopoietic growth factors and interleukins IL-3, IL-4, IL-5, IL-9, and GM-CSF, and, the EGR-1 transcription factor (Huebner et at., Science 230: 1282-1285 (1985); Le Beau et al., Science 231: 984-987 (1986) and ibid. (1989); Sutherland et al., Blood, 71: 1150-1152 (1988); Warrington et at., Genomics 13: 803-808 (1992)). However, none of these genes currently appear to fulfill the requirements expected of a candidate tumor suppressor gene. Loss of one IL-3, IL-4, IL-5, and GM-CSF allele has been frequently, though not consistently, reported in leukemia and MDS patients with del(5q) (Le Beau et at., ibid. (1986), Proc. Natl. Acad. Sci. USA 84: 5913-5917 (1987), ibid. (1989); Nimer and Golde, ibid. (1987)). However, no reduction to homozygosity, structural rearrangements, or mutations in the residual alleles have been discovered (see Nimer and Golde, ibid. (1987)). Recent studies of EGR-1 in del(5q) patients have yielded similar negative findings (G. Gilliland et at., Harvard University, personal communication). Thus, a candidate tumor suppressor gene remained to be identified in this region.